Abstract The ventral tegmental area (VTA) sends dense projections to the nucleus accumbens (NAc), constituting the backbone of the mesolimbic system. The mesolimbic system is often targeted by drugs of abuse, stress, and other severe experience to change the emotional and motivational states, resulting in a variety of psychological and psychiatric disorders. In addition to dopamine and GABA, the VTA-to-NAc projections also release glutamate. The VTA-to-NAc glutamatergic transmission has been thought to critically regulate NAc principle medium spiny neurons (MSNs), and contribute to motivated behaviors as well as behavioral alterations after exposure to drugs of abuse. However, despite extensive exploration, only weak cellular effects of the VTA-to- NAc glutamatergic signaling have been detected. For example, VTA-released glutamate evokes EPSCs in NAc MSNs as well as in interneurons, but these EPSCs are usually very small compared to EPSCs evoked from other major glutamatergic sources. A critical question is whether there are cellular mechanisms through which the VTA-to-NAc glutamatergic transmission effectively regulates NAc MSNs and NAc-based behaviors. Our preliminary results reveal a novel and robust cellular role of the VTA-to-NAc glutamatergic projection. Specifically, activation of the VTA-to-NAc glutamatergic projection transiently inhibited the responsiveness of MSNs to other major excitatory inputs, providing a potential time-locked shunting of NAc MSNs upon VTA activation. Thus, rather than a depolarization driver, the VTA-to-NAc glutamatergic projection functions as a regulator of other ongoing excitatory inputs to NAc MSNs. This regulation can be particularly important in behaving animals, in which activation of NAc MSNs must be finely controlled in response to incoming excitatory inputs to achieve select behaviors. Our subsequent preliminary results suggest a novel mechanism mediating this regulation, a mechanism involving activation of NAc astrocytes and astrocytic release of gliotransmitters. Because each astrocyte ensheathes a population of neurons, this potential effect of astrocytes may help synchronize population activities of NAc MSNs. The objective of this application is to thoroughly characterize the cellular basis of this VTA-to-NAc glutamatergic transmission-mediated regulation of NAc MSNs. Based on extensive preliminary results, we hypothesize a neuron-glia-neuron interaction mechanism: activation of the VTA-to-NAc glutamatergic projection activates metabotropic glutamate receptor 5 (mGluR5) on NAc astrocytes, which results in astrocytic release of ATP to presynaptically inhibit ongoing excitatory synaptic transmissions to NAc MSNs from other major glutamatergic inputs. By testing this hypothesis, we expect to characterize the cellular and circuitry mechanisms underlying VTA-to-NAc glutamatergic regulation. These mechanisms and involved molecular substrates may provide a conceptual and experimental foundation for future comprehensive studies to reveal the cellular, circuitry, and behavioral roles of VTA-to-NAc glutamatergic signaling, and how to manipulate this signaling to achieve clinical benefits.